JP2022546398A - Systems and methods for processing reproductive specimens - Google Patents

Abstract

a plate for supporting an analyte system comprising a container and an analyte contained therein; a camera disposed above the plate and configured to generate an image of the analyte system; a light source located below the plate to illuminate the light; a light block to block a portion of the light from reaching the sample system to provide dark field illumination of the sample at the camera; and one or more processors coupled to and configured to track the position of a sample within a sample container during a sample processing protocol based on images.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of prior US Provisional Patent Application No. 62/894,202, filed Aug. 30, 2019, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD The present disclosure relates to sample processing systems, such as automated vitrification systems, and methods for tracking the position of samples within sample containers undergoing sample processing protocols in such sample processing systems.

BACKGROUND Cryopreservation containers are used in the field of assisted reproductive technology (ART) to store and preserve live reproductive specimens (eg, oocytes, embryos, and blastocysts). Cryopreservation refers to the process by which specimens are preserved for extended periods of time by cooling to sub-zero temperatures. For example, a cryopreservation container can contain and support a specimen that has undergone vitrification; vitrification is the process of transferring a substance from a liquid phase to a solid phase (e.g., glass) without the formation of ice crystals within the cells of the specimen. to rapidly transition to

A typical protocol for vitrifying a reproductive specimen includes the following steps: exposing the specimen to multiple treatment solutions according to a detailed vitrification protocol; subsequently transferring the specimen to a cryopreservation container; The cryopreservation container containing the specimen is then exposed to a cold coolant (e.g., liquid nitrogen) to rapidly cool the specimen's cells to a glassy state before ice crystals can form within the cells. stage to do. The cryopreservation container can be stored in a cryogen until the specimen is ready for use in a reproductive procedure.

Overview Generally, the present disclosure relates to a specimen processing system that can be used to prepare biological specimens for cryopreservation in specimen containers in an automated fashion according to specimen processing protocols (e.g., vitrification protocols).

In one aspect, a sample processing system includes a plate for supporting a sample system, the sample system including a container and a sample contained therein. a camera disposed above the plate and configured to generate an image of the specimen system; a light source disposed below the plate for directing light toward the plate; a light stop for blocking a portion of the light from reaching the specimen system to provide darkfield illumination of the specimen; electronically coupled to the camera and during the specimen processing protocol based on the image; and one or more processors configured to track the position of the specimen within the specimen container.

Aspects may include one or more of the following features.

In some embodiments, the sample processing system further includes an adjustable lens for focusing light onto the sample system.

In some aspects, the sample processing system further includes a processing station that positions the camera.

In some aspects, the processing station defines a receptacle adjacent the plate for positioning a sample container.

In some aspects, the processing station includes a mount for selectively positioning a camera at the processing station.

In some aspects, the sample processing system further includes a rotatable platform to which the processing station is fixed for applying a centripetal force to the sample to induce motion in the sample within the sample container.

In some embodiments, the one or more processors are further configured to convert the image from color to grayscale.

In some embodiments, the one or more processors are further configured to remove noise from the image.

In some embodiments, the one or more processors are further configured to detect an object corresponding to the analyte within the image.

In some embodiments, the one or more processors are further configured to determine parameters including one or more of position, velocity, and direction of the specimen as it moves within the specimen container.

In some aspects, the one or more processors are configured to output one or more of the parameters.

In some embodiments, the sample processing system further includes a motor capable of adjusting movement of the rotatable platform based on one or more of the parameters.

In some embodiments, a light block blocks a portion of the light from reaching the central axis of the sample container so that the edge of the sample remains visible to provide darkfield illumination at the camera. are arranged as follows.

In some embodiments, the light source includes multiple light emitting diodes.

In some embodiments, the camera is configured to scan an identification label on a specimen container.

In some embodiments, the one or more processors are configured to track positions of each of a plurality of specimens within a specimen container based on images during a specimen processing protocol.

In some aspects, the sample processing system further includes a vibrating assembly configured to direct movement of the sample within the sample container during a sample processing protocol.

In some embodiments, the sample processing system further includes a cutting station configured to cut and release the distal portion of the sample container with the sample contained therein after completion of the sample processing protocol.

In some embodiments, the specimen is a reproductive specimen.

In some embodiments, the specimen processing protocol includes a vitrification protocol.

In another aspect, a method of processing a sample in a sample container includes the steps of: generating an image of the sample in the sample container with a camera positioned above a plate supporting the sample container. directing light toward the plate from a light source located below the plate; blocking a portion of the light from reaching the sample with a light block to provide dark field illumination of the sample at the camera; and tracking the position of the specimen within the specimen container based on the images in one or more processors in electronic communication with the camera during a specimen processing protocol.

Aspects may include one or more of the following features.

In some embodiments, the disclosed method further comprises focusing the light onto the specimen with an adjustable lens.

In some aspects, the method further includes positioning the camera at the processing station.

In some embodiments, the method further comprises positioning the specimen container within a receptacle of the processing station adjacent to the plate.

In some aspects, the method further includes selectively positioning a mount that supports the camera at the processing station.

In some embodiments, the method further includes applying a centripetal force to the specimen to induce movement in the specimen within the specimen container by rotating a platform to which the processing station is fixed.

In some embodiments, the method further comprises converting the image from color to grayscale at the one or more processors.

In some embodiments, the method further comprises removing noise from the image at said one or more processors.

In some embodiments, the method further comprises detecting, at said one or more processors, an object corresponding to the analyte within the image.

In some embodiments, the method comprises determining, at the one or more processors, parameters including one or more of position, velocity, and direction of the specimen as it moves within the specimen container. Including further.

In some embodiments, the method further comprises outputting one or more of the parameters from the one or more processors.

In some embodiments, the method further comprises adjusting movement of the platform with the motor based on one or more of the parameters.

In some embodiments, the method includes blocking a portion of the light from reaching the central axis of the sample container so that the edge of the sample remains visible to provide darkfield illumination at the camera. further includes

In some embodiments, the light source includes multiple light emitting diodes.

In some embodiments, the method further comprises scanning the specimen container identification label with a camera.

In some embodiments, the method further comprises tracking, at the one or more processors, the position of each of a plurality of specimens within a specimen container based on the images during a specimen processing protocol.

In some embodiments, the method further comprises directing movement of the specimen within the specimen container with the vibrating assembly during the specimen processing protocol.

In some embodiments, the method further includes, after completion of the sample processing protocol, cutting and releasing the distal portion of the sample container with the sample contained therein at a cutting station.

In some embodiments, the specimen is a reproductive specimen.

In some embodiments, the specimen processing protocol includes a vitrification protocol.

Aspects may provide one or more of the following advantages.

In some embodiments, the sample processing system includes one or more processing stations that can be configured with multiple mounting and supporting components for precisely positioning sample containers as desired. The sample processing system also advantageously has a microcontroller that can adjust the rotational speed of the platform on which the sample container rotates and the duration of one or more phases of the sample processing protocol based on feedback from the vision system. Also includes

For example, in some embodiments, a vision system located at each processing station is configured to provide dark field illumination of the specimen for optimal visualization and tracking of the specimen during the specimen processing protocol. The configuration and functionality of the various components of the vision system to achieve darkfield illumination advantageously allow for precise control and constraint of the intensity, exposure time, and wavelength of light emitted from the light source to the specimen. , which can be important for the viability of delicate biological specimens.

Further, in some embodiments, the cameras of the vision system continuously generate images of the specimen, and the images are processed by a computing device executing software algorithms that process the images to track the position of the specimen. The linear motion of the specimen may be tracked in real time throughout the specimen processing protocol by feeding it as a real-time video feed. Based on feedback from software algorithms, the microcontroller advantageously controls the rotational speed, spin direction, and acceleration of the platform so that the specimen is exposed to a substantially constant centripetal force as programmed by the user. good. Such protocol adjustments may optimize the exposure time of the specimen to the processing medium.

1 is a front perspective view of a sample processing system that may be used to prepare samples disposed within sample containers; FIG. FIG. 2 is a side perspective view of the sample processing system of FIG. 1; FIG. 2 is a rear perspective view of the sample processing system of FIG. 1; 2 is a top perspective view of the sample processing system of FIG. 1 with certain components of the processing station omitted; FIG. 2 is a side view of a sample container that may be processed in the sample processing system of FIG. 1; FIG. 6 is a cross-sectional view of the proximal end region of the specimen container of FIG. 5 including an identification (ID) label provided as an RFID tag; FIG. 6 is a cross-sectional view of the proximal end region of the specimen container of FIG. 5, including an ID label provided as a barcode tag; FIG. 6 is a cross-sectional view of the proximal end region of the specimen container of FIG. 5, including an ID label provided as a QR code tag; FIG. 2 is a front perspective view of the sample processing system of FIG. 1 with certain portions of the housing omitted to expose certain internal components; FIG. 2 is a bottom perspective view of the sample processing system of FIG. 1 with certain portions of the housing omitted to expose certain internal components; FIG. 2 is a front perspective view of the platform and certain other related components of the sample processing system of FIG. 1; FIG. FIG. 12 is a top perspective view of the platform of FIG. 11; 2 is an exploded perspective view of the vision system of the sample processing system of FIG. 1; FIG. 2 illustrates a series of movements of a sample within the sample container of FIG. 1 for processing the sample according to the protocol performed in the sample processing system of FIG. 1; 2 illustrates a series of movements of a sample within the sample container of FIG. 1 for processing the sample according to the protocol performed in the sample processing system of FIG. 1; 2 illustrates a series of movements of a sample within the sample container of FIG. 1 for processing the sample according to the protocol performed in the sample processing system of FIG. 1; 2 illustrates a series of movements of a sample within the sample container of FIG. 1 for processing the sample according to the protocol performed in the sample processing system of FIG. 1; 2 illustrates a series of movements of a sample within the sample container of FIG. 1 for processing the sample according to the protocol performed in the sample processing system of FIG. 1; 2 illustrates a flow chart of a software algorithm for processing images of specimens during a protocol performed in the specimen processing system of FIG. 1; 1 is a perspective view of a vibrating assembly of sample processing system 100. FIG. FIG. 10 is a side view of a cutting and sealing station of the sample processing system; FIG. 3B is a side view of a cutting station of the sample processing system;

DETAILED DESCRIPTION FIGS. 1-4 illustrate a specimen processing system 100 that can be used to prepare a biological specimen for cryopreservation in a specimen container 1000 in an automated fashion according to a specimen processing protocol (eg, a vitrification protocol). illustrate various drawings of. Referring to FIG. 5, a specimen 1001 is placed in a specimen container 1000, and the specimen container 1000 is placed in a cryogenic substance (e.g., a liquid It is designed for cryopreservation and cryopreservation of specimens 1001 in a viable and vitrified state in nitrogen, cryogenic plasma, or liquid helium. Specimen 1001 may be a single cell, a collection of free (eg, non-attached) cells, or a collection of attached cells (eg, multicellular tissue). The specimen 1001 can be a reproductive specimen (e.g., sperm cells, oocytes, zygotes, blastocysts, gastrula, or embryos) or a non-reproductive specimen (e.g., one or more T cells or blood cells). There may be. Specimen 1001 may be a mammalian tissue sample or a non-mammalian tissue sample. In some embodiments, specimen 1001 may be an agricultural specimen such as canola. In other examples, analyte 1001 may be a non-biological analyte, such as various chemicals or other non-biological analytes.

The sample processing system 100 and sample container 1000 are jointly designed to exploit the mass properties (eg, density and fluid dynamics) of the sample 1001 against the mass properties of various process media. Specimen container 1000 is thus provided as an elongated tube 1002 internally preloaded with a plurality of fluids to which specimen 1001 is exposed during cryopreservation. In particular, the specimen 1001 may be moved axially 1003 within the specimen container 1000 by centrifugal forces acting on the specimen 1001 within the processing system 100, as described in greater detail below.

Elongated tube 1002 may be hermetically sealed with proximal and distal closures 1004,1006. In some embodiments, elongated tube 1002 contains an equilibration solution 1008 (eg, a relatively low density cryoprotectant) and a vitrification solution separated by a separation fluid 1012 (eg, air bubbles or immiscible media). 1010 (eg relatively high density cryoprotectant). Such separation of the equilibration solution 1008 and the vitrification solution 1010 facilitates proper handling of the specimen 1001 during the vitrification protocol (e.g., placing the specimen 1001 in a particular solution sequentially for a desired period of time). exposure). In some embodiments, the elongated tube 1002 has a proximal air pocket 1014 that separates the equilibration solution 1008 from the proximal closure 1004 and a vitrification solution 1010 that separates the distal closure 1006 (e.g., from the elongated tube 1002). A distal air pocket 1016 (which occupies a portion of the interior volume of tapered portion 1018) is also preloaded.

Elongated tube 1002 is a thin capillary tube of very small diameter (eg, having an inner diameter on the order of 10 ⁻⁴ m). Elongated tube 1002 has a substantially constant diameter along main portion 1020 (eg, a cylindrical portion) and a varying diameter that gradually decreases along tapered portion 1018 . The lumen of elongated tube 1002, at its smallest internal diameter, is large enough to accommodate specimen 1001; specimen 1001 typically has a diameter or width within the range of about 50 μm to about 150 μm. Specimen container 1000 typically has an overall length of about 15 mm to about 260 mm (eg, about 150 mm). Elongate tube 1002 is typically made of one or more materials that are transparent or translucent to allow visualization of analyte 1001 contained within elongate tube 1002 and are resistant to cryogenic substances. Examples of materials from which elongate tube 1002 may be made include polymers such as polystyrene, polypropylene, polyvinyl acetate, and polycarbonate, as well as fluoropolymers.

6-8, specimen container 1000 further includes an identification (ID) label 1022, 1024, or 1026 attached to elongated tube 1002 near proximal closure 1004, respectively. The ID label may be attached to the elongated tube 1002 with an adhesive sticker or embedded within the wall of the elongated tube 1002 . The ID label contains machine readable information and may additionally contain human readable information written on the outer surface of the ID label. Either or both of the machine-readable and human-readable information may include various patient data such as name, date of birth, unique reference code (eg, alphanumeric string), and other patient data. The ID label on the sample container 1000 may be detected and read by the scanning component of the sample processing system 100, as described in more detail below. As shown in FIGS. 6-8, respectively, the ID label may be a radio frequency identification (RFID) tag 1022 (eg, including an internal antenna), a bar code 1024 tag (eg, including one-dimensional code format), or a tag (eg, including two-dimensional code). may be embodied as a quick response (QR) code 1026 tag (including dimension code format).

1, 2, and 4, the sample processing system 100 includes a plurality of processing stations 102 to which each sample container 1000 may be secured for performing a sample processing protocol, along which the processing stations 102 are arranged. A console is provided that includes a platform 104 that rests on the platform 104 , a housing 106 that encloses internal components located below the platform 104 , a handle 188 for lifting or otherwise moving the sample processing system 100 , and a lid 108 . Lid 108 can be opened from housing 106 to allow access to processing station 102 and can be closed over housing 106 to prevent access to or otherwise protect processing station 102 . The sample processing system 100 has a display screen 110 positioned along the front wall of the housing 106 that presents various user interfaces and sets various operating parameters of the sample processing system 100 and various processing parameters of sample protocols. a power switch 192; a cable port 114; and a power connector 116 positioned along the rear wall of the housing 106.

Housing 106 is designed to rest on a table top, floor, or another flat surface. Housing 106 defines an air vent 118 positioned along the side wall and an air vent 120 positioned along the rear wall. Air vent 118 allows air to circulate into and out of housing 106 to prevent internal components disposed within housing 106 from exceeding a threshold temperature of approximately 80 degrees Celsius. Housing 106 also defines a power connector 122 along the rear wall. Housing 106 is connected to lid 108 via hinge 124 .

In some embodiments, the housing 106 and lid 108 of the sample processing system 100 together have an overall length of about 0.2 m to about 1.0 m, an overall width of about 0.2 m to about 1.0 m, and a width of about 0.2 m to about 1.0 m. with a total height of m. In some embodiments, the sample processing system 100 has a weight within the range of about 5 kg to about 50 kg, and typically has an ambient temperature of about 18°C to about 28°C. Stored on the floor, the floor of a storage facility, a table, or a countertop. In some embodiments, receptacle 162 of processing station 102 has a length of about 5 cm to about 15 cm and a width of about 1 cm to about 5 cm. Housing 106 and lid 108 are typically made of materials that provide a significant degree of thermal insulation, such as polymers.

Additionally, the sample processing system 100 includes a timer 126 for tracking the duration of various phases of the sample processing protocol, a reading component 128 programmed to read the ID label of the sample container 1000, and a and a microcontroller 130 programmed to control various features and functionality. Timer 126, readout component 129, and microcontroller 130 (all shown schematically in FIG. 1) may be located in positions suitable for their respective functions. For example, any of timer 126, read component 129, and microcontroller 130 may be mounted on any sidewall (eg, base, side, top, or bottom) of housing 106, or on a support member attached thereto. may For example, in some embodiments, microcontroller 130 may be located adjacent display screen 110 .

Display screen 110 allows a user to enter a number of parameters that govern the operation of sample processing system 100 to process (eg, vitrify) one or more samples 1001 . In some embodiments, such input parameters relate to specimen 1001, such as the developmental stage of specimen 1001 (eg, resulting in selection of oocyte or blastocyst protocol, etc.). Display screen 110 may be an integrated touch screen or may be a touchless screen associated with tactile control elements such as buttons, knobs, or dials.

Microcontroller 130 includes one or more processors in communication with and/or programmed to control various actuators and sensors of sample processing system 100 related to various automated features; Such features include receiving and instantiating user selection inputs at display screen 110; reading the ID label of sample container 1001; executing timer 126; spinning the platform 104; detecting the open or closed state of the lid 108; and providing audible and/or visual feedback regarding the progress of the sample processing protocol; In some embodiments, platform 104 can be activated to spin only after lid 108 is closed and interlocked with housing 106 . Additionally, if the platform 104 is spinning as part of the sample processing protocol, the lid 108 may not be able to be opened until the platform 104 stops spinning.

Referring specifically to FIG. 4, platform 104 defines a plurality (eg, six) of slots 132 that allow processing station 102 to be secured (eg, bolted) to platform 104 in fixed positions. Slot 132 is formed as an elongated opening along which sample container 1000 may be aligned, thus defining a plurality of arbitrary positions at which sample container 1000 may be positioned on platform 104 . Flanking each slot 132 are located a set of four holes 134 and two sets of two holes 136 distributed in a parallel arrangement to the slot 132 . Accordingly, the processing station 102 can be attached to the platform 104 at holes 134 , 136 to interrogate a specimen 1001 inside a specimen container 1000 positioned along the slot 132 . The placement of the multiple slots 132, the size of the various components of the processing station 102, and the functional requirements of the sample processing system 100 (e.g., maintaining a substantially balanced mass across the platform 104 during the protocol). Due to this, in some embodiments, only two or three processing stations 102 may be installed on the platform 104 at any given time, and the two or three processing stations 102 may be installed on the platform 104. They should be circumferentially spaced substantially equally apart from each other around. In other embodiments, different numbers of processing stations 102 and their spacing may be implemented, so long as a method of balancing mass across the platform 104 is used, such as strategically placing counterweights along the platform 104. .

9 and 10, where certain portions of housing 106 and lid 108 have been omitted to expose certain internal features, sample processing system 100 includes a printed circuit board (PCB) assembled with platform 104. ) 138 and motor assembly 140, and PCB 154 positioned along the front wall of housing 106 (omitted in FIGS. 9 and 10 for clarity). In some embodiments, timer 126 and microcontroller 130 (shown schematically in FIG. 1) are implemented on PCB 154 . The assembly of platform 104 and motor assembly 140 ensures that acceleration occurs quickly and smoothly during changes in rotational speed of platform 104 .

Referring specifically to FIG. 10, PCB 138 is attached (eg, bolted) to the bottom surface of platform 104 and is sized, positioned, and oriented to align with multiple slots 132 of platform 104 (eg, 6 slots). ) extension plate 142 . A matrix (eg, two arrays) of multiple light emitting diodes (LEDs) 144 are mounted on the top surface of each extension plate 142 and exposed through slots 132 in platform 104 (see FIG. 4). Motor assembly 140 includes a rotatable motor block 190, a support plate 146 attached to the top surface of motor block 190, posts 148 extending from support plate 146 to platform 144, and a support plate 148 extending from motor block 190 (e.g., support plate 146). and a cylindrical coupling unit 150 extending to the platform 104 (through). Motor block 190 is capable of executing specific commands to move platform 104 to specific desired positions to perform various actions (e.g., mounting or dismounting sample container 1000 from sample processing system 100). It may be a servo or stepper motor fitted with encoders to provide continuous monitoring of motor speed and position. Cylindrical linkage unit 150 is attached to both platform 104 and motor block 190 such that rotation of motor block 190 causes rotation of linkage unit 150 and platform 104 about central axis 152 of platform 104. . In addition, sample processing system 100 also converts source electricity (eg, 110 volts/220 volts) for motor power and heat sink 196 and some or all of the components within sample processing system 100 that require electricity. and power converter 198 . Cylindrical coupling unit 150 includes a plurality of cylindrical electrical contacts 156 (eg, slip rings) that transmit data and control signals between processing station 102 , motor block 190 and microcontroller 130 .

11 and 12, each processing station 102 includes a lower bracket 158 and an upper bracket 160 that together define a receptacle 162 for holding a specimen container 1000 along slot 132 of platform 104. As shown in FIG. In some embodiments, processing station 102 further includes one or more spring-loaded retaining strips or clamps that help secure specimen container 1000 within receptacle 162 . Lower bracket 158 is positioned to align with one or more of holes 134 and one or more of holes 136 to mount processing station 102 to platform 104 along a particular slot 132. 164 and holes 166 are defined. Lower bracket 158 also defines a plurality (eg, four) of flanges 168 that secure upper bracket 160 to lower bracket 158 . Each processing station 102 also includes a post 170 that passes through alignment holes defined by the upper and lower brackets 160, 158, respectively, to ensure that the upper bracket 160 is properly positioned along the lower bracket 158. Lower bracket 158 also defines opposed raised slots 176 and lateral through channels 178 . Upper and lower brackets 160, 158 and platform 104 of processing station 102 are typically made of one or more metals such as aluminum, magnesium, stainless steel, and other metals.

Each processing station 102 includes a camera 180 by which movement of the specimen 1001 within the specimen container 1000 can be observed, a mounting bracket 182 supporting the camera 180, and a cover plate for containing the camera 180 within the mounting bracket 182. 194 and further including. Mounting bracket 182 has two opposed elongated protrusions sized and positioned to slide within raised slot 176 to position camera 180 in a desired position along lower bracket 158. Section 184 is defined. Mounting bracket 182 further includes two sets of opposed holes 186 along protrusion 184 that can be selectively aligned with through channels 178 to secure mounting bracket 182 to bottom bracket 158 in a desired location. stipulate.

When specimen 1001 is processed in specimen container 1000 in specimen processing system 100, detailed information about specimen 1001 may be entered by an operator at display screen 110, or such information may be received from another device through a data connection. It may be automatically imported into the sample processing system 100 . In some embodiments in which the sample container 1000 is not pre-populated with an ID label (e.g., ID label 1022, 1024, or 1026), the sample processing system 100 uses automatically imported information to generate human-readable information or It may be configured to print the barcode on an ID label and then attach the ID label to the sample container 1000, or the printed ID label may then be manually attached to the sample container 1000 by an operator. good too.

In either case, once the details about the specimen 1001 have been manually entered or automatically imported, the operator then loads the specimen container 1000 with the ID label into the receptacle 162 at the processing station 102. do. A reading component 128 may detect the presence of specimen container 1000 in receptacle 162 by reading the ID label, and may communicate such detection to microcontroller 130 . In some embodiments, reading component 128 may be a feature of camera 180 . For example, if ID labels are provided as barcode labels 1024 or QR code labels 1026, camera 180 may be configured and programmed to read such labels.

If the manually entered or automatically imported information does not match the information read from the ID label by the reading component 128, the sample processing system 100 generates and displays an error on the display screen 110 and displays the sample Prevent activation of processing protocols. If the manually entered or automatically imported information matches the information read from the ID label by reading component 128, sample processing system 100 initiates timer 126 to process sample 1001 according to the specified protocol. may be activated. In accordance with one or more signals received from microcontroller 130, platform 104 is configured to move specimen 1001 within specimen container 1000 along axial direction 1003 toward distal closure 1006 according to protocol (see FIG. 5). To apply sufficient centripetal force to specimen 1001, it may be spun around central axis 152. FIG. While the platform 104 is spinning, the specimen 1001 and various processing media (e.g., equilibration and vitrification solutions 1008, 1010, and any other media) within the specimen container 1000 are visualized (e.g., imaged) by the camera 180. ) may be In some embodiments, one or more parameters of the protocol are determined by or associated with the type of ID label (e.g., RFID, barcode, or QR code) present on the specimen container 1000. good.

As will be described in more detail below, microcontroller 130 controls the rotational speed of platform 104 and the duration of one or more phases of the protocol based on feedback from a vision system (eg, including camera 180) regarding the axial position of specimen 1001. You may adjust either or both of and. Such protocol adjustments may optimize the sample exposure time to the process medium within sample container 1000 . Once the processing protocol is completed, the specimen container 1000 may be removed from the receptacle 162 and the specimen 1001 contained within the specimen container 1000 may be placed in cryogenic material for vitrification and cryopreservation.

As noted above, camera 180 may be used to track the position of specimen 1001 within specimen container 1000 during a specimen processing protocol. As shown in FIG. 13, each camera 180 is a component of the vision system 200 located at each processing station 102 of the sample processing system 100 . Each vision system 200 includes, in addition to a camera 180, an optically clear plate 202 on which the specimen container 1000 can be supported; upper and lower lenses 204, 206 (eg, plano-convex lenses); , 206; two compression springs 210 each surrounding the adjusting screw 208; an extension plate 142 of the PCB 138 and a matrix of LEDs 144 distributed along the extension plate 142 (e.g. and a centrally directed light beam (e.g., a light beam directed substantially toward the central axis 1028 of the sample container 1000) directed toward the sample container 1000. It further includes an opaque light block 214; which blocks it from entering. In some embodiments, plate 202 of vision system 200 may be placed within slot 132 of platform 104 . Camera 180 is typically positioned above platform 104 at a distance of about 1 cm to about 5 cm.

Upper and lower lenses 204, 206 collimate the light emitted from LED 144 into a light beam and direct the light beam onto the expected path of specimen 1001 (e.g., approximately central axis 1028 of specimen container 1000). ) is a focusing lens that can be focused. Therefore, the adjustment screw 108 and the compression spring 210 therearound are arranged to adjust the upper and lower lenses 204, 206 so that the focal point of the light beam coincides with the height of the support plate 202 on which the specimen container 1000 is held. allows you to adjust the height of the Support plate 202 is typically positioned above light source 212 at a distance of about 0.1 cm to about 1.5 cm.

Shade 214 is a light beam directed toward the center from LED 144 such that the peripheral (e.g., off-axis) edge of specimen 1001 is illuminated when top and bottom lenses 204, 206 are properly focused. to block Thus, the periphery of specimen 1001 appears brighter than the interior regions of specimen 1001, making specimen 1001 more visible to camera 180, in a manner similar to darkfield illumination. In addition, vision system 200 may include filtering functionality to block light with wavelengths less than about 500 nm from reaching specimen 1001; health and subsequent biological development. Thus, the configuration and functionality of the various components of vision system 200 to provide darkfield illumination are to precisely control and constrain the intensity, exposure time, and wavelength of light emitted from light source 212 onto specimen 1001. , which can be important for the viability of delicate biological specimens 1001 .

Camera 180 continuously generates images of specimen 1001 and feeds the images at regular intervals or in the form of a real-time video feed to monitor linear motion of specimen 1001 throughout the specimen processing protocol. Tracking may be in real-time; the feed is wireless to a remote computing device running a software algorithm 300 (see FIG. 19) that processes the images to track the position of the specimen 1001. or through a hardwired connection via electrical contacts 156 to one or more processors of microcontroller 130 running software algorithm 300 . Referring to FIG. 14, the sample container 1000 remains in place (e.g., stationary) within the receptacle 162 for a first predetermined exposure time during the sample processing protocol so that the sample 1001 can equilibrate in the equilibration solution 1020. ) is left undisturbed. The first exposure time may range from about 5 minutes to about 15 minutes, depending on various parameters of a typical ART protocol.

During the first exposure time, the equilibration solution 1020 draws water molecules from the specimen 1001 and injects cryoprotectant into the specimen 1001 according to the osmotic potential. The reduction in water content and the addition of cryoprotectants help minimize damage to the cellular components of specimen 1001 during freezing and warming cycles. Analyte 1001 is denser than equilibration solution 1020 and therefore descends very slowly through equilibration solution 1020 due to gravity over time, but as shown in FIG. It is suspended in solution 1020 and has not reached separation fluid 1024 by the end of the first exposure time.

15-18, once the analyte 1001 has been exposed to the equilibration solution 1020 for the predetermined exposure time, the equilibration solution 1020 and the analyte 1001 are advanced axially through the separation fluid 1024 to the vitrification solution 1022. Therefore, the platform 104 is activated to spin the sample container 1000 at the selected slow speed. Specimen container 1000 is typically spun at an angular velocity of about 50 rpm to about 1200 rpm for about 0.5 minutes to about 5 minutes; It exerts a centripetal force on the specimen 1001 that is sufficient, but not sufficient to cause mechanical damage to the specimen 1001 . Such speeds (e.g., corresponding to about 5 g to about 200 g) are much lower than those of conventional laboratory centrifuges at very low speeds; conventional laboratory centrifuges typically The specimen can be spun around the centrifugal axis at speeds within the range of about 4000 rpm to about 300,000 rpm (eg, corresponding to about 2,500 g to about 65,000 g).

Referring specifically to FIG. 15, during the first phase of spinning, the analyte 1001 descends in the equilibration solution 1020, while the equilibration solution 1020 containing the analyte 1001 descends through bulk motion and is used for separation. Through fluid 1024 (eg, thereby displacing separating fluid 1024) to vitrification solution 1022. Referring specifically to FIG. 16, during the next phase of spinning equilibration solution 1020 reaches vitrification solution 1022 and analyte 1001 passes from equilibration solution 1020 into vitrification solution 1022 . Referring particularly to FIG. 17, during the next phase of spinning, equilibration solution 1020 merges with vitrification solution 1022 to form a composite vitrification solution 1030 (e.g., equilibration solution 1020, vitrification solution 1022, and equilibration solution 1020). (including a mixed solution interface layer between solution 1020 and vitrification solution 1022 ), and specimen 1001 continues to descend through composite vitrification solution 1030 .

Referring specifically to FIG. 18, during the final phase of spinning, surface tension causes the specimen 1001 to rest on the meniscus 1032 of the distal air pocket 1028, thus avoiding contact with the relatively hard walls of the elongated tube 1002. FIG. For example, due to the balance between the surface tension at the interface of composite vitrification solution 1030 and distal air pocket 1028 and the tension between composite vitrification solution 1030 and the inner wall of tapered portion 1016, distal air pocket 1016 is not sufficient to break the meniscus 132. Therefore, specimen 1001 cannot penetrate meniscus 1032 .

With the specimen 1001 resting on the meniscus 1032 of the distal air pocket 1028 at the completion of the spin, the timer 126 is started and a second predetermined exposure time for exposing the specimen 1001 to the composite vitrification solution 1030. Specimen container 1000 remains in place (eg, stationary) within receptacle 162 throughout. The second exposure time may range from about 0.5 minutes to about 2 minutes, depending on various parameters of a typical ART protocol. During the second exposure time, water in specimen 1001 is displaced by permeation of cryoprotectant in composite vitrification solution 1030 into specimen 1001, thereby dehydrating the specimen and removing cryoprotectant from specimen 1001. is injected further. Such a staged progression of medium concentrations avoids situations where the initial osmotic pressure difference is too large; if the initial osmotic pressure difference is too large, water will flow out of the cells at a faster rate than the cryoprotectant can enter the cells. As the particles exit, the cells of specimen 1001 may shrink too much and shrink too quickly.

Multiple accesses to containers containing specimens are made to deliver or remove different treatment media, or specimens are transferred to different containers (e.g. Petri dishes, test tubes, or flasks) during the ART process. In some cases, equilibration solution 1020 and vitrification solution 1022 may suffer from contamination, mechanical damage (eg, by micropipettes or other specimen retention or fluid delivery devices), or other accidental mishandling. are preloaded within the specimen container 1000 to prepare the specimen 1001 for vitrification within a single isolated environment (e.g., the lumen of the specimen container 1000) without possible exposure to those be able to.

In some embodiments, once the second exposure time has expired, the specimen container 1000 containing the specimen 1001 is then removed from the receptacle 162 into a long-term cryogenic condition capable of maintaining the specimen 1001 in cryogenic conditions for periods of up to about 20 years. Manually transferred to a cold storage structure. In some cases, specimen container 1000 may be stored in a long-term cryogenic storage structure for much shorter periods of time (eg, as short as a few hours).

The software algorithm 300 used to track the position of the specimen 1001 may run on the microcontroller 130 or may be sent to the specimen processing system via a data connection (eg, USB connection, RS232 connection, or wireless data connection). It may run on a separate external computing device (eg, a desktop computer, laptop, tablet, or single board computer) running an operating system electrically coupled to 100 . Referring to FIG. 19, the software algorithm 300 enters a processing flow loop where the software acquires (302) a single color image from the camera feed, converts that image to grayscale, and converts each pixel of the grayscale image to Store 304 the grayscale image in an array that holds the gray values for . Algorithm 300 then defines the size and position of the area of interest relative to the field of view of camera 180 for detecting the edges (e.g. contours) of specimen container 1000 and thereby tracking the position of specimen 1001. To do so, an edge detection routine is performed on the grayscale image (306).

The algorithm 300 then captures (308) a first subsequent color image from the camera feed, waits (310) for a period of time, and then captures (312) a second subsequent color image from the camera feed. The first and second subsequent color images are cropped to the area of interest as they are captured. Algorithm 300 also converts the first and second subsequent color images to grayscale, and stores the first and second grayscale images in an array that holds a gray value for each pixel in the grayscale image. (314). Algorithm 300 then compares the first and second grayscale images to each other and generates an additional array that stores the pixilation difference between the first and second grayscale images as a difference image. (316). Algorithm 300 converts the luminous intensity data from the difference image to binary values based on the upper and lower constraints to generate a first binary thresholded difference image (318). For example, all image data that falls between the upper and lower constraints are retained in the first binary threshold difference image, while all image data that does not fall within the range defined between the upper and lower constraints. Image data is rejected.

Algorithm 300 then blurs the first binary threshold difference image to remove noise, thereby producing a first blurred difference image (320). Algorithm 300 again converts the luminous intensity data from the first blurred difference image to binary values based on the upper and lower constraints to provide a second binary threshold with less noise compared to the first binary threshold difference image. A difference image is generated (322). In this case, the binary value, upper constraint, and lower constraint are independent of what was used to generate the first binary threshold difference image. Algorithm 300 also blurs the second binary threshold difference image to further remove noise, thereby producing a second blurred difference image (324).

The algorithm then passes (326) the second binary threshold difference image to an object detection routine; in this routine the specimen 1001 may be identified within the image. If no specimen 1001 is identified in the image (328), algorithm 300 returns to capturing the first subsequent color image from the camera feed (308). Once the specimen 1001 is identified in the image (328), the algorithm 300 categorizes (eg, determines) the position of the specimen 1001 and stores the position in an array of object positions (330). Algorithm 300 identifies specimens 1001 based on the number of pixels (eg, for a known camera resolution) using predetermined maximum and minimum thresholds; Represents a circular area (332). Algorithm 300 stores the specimen 1001 center position, velocity (based on the time elapsed between previous processed images and the position of specimen 1001 in the current and previous processed images), and orientation in a separate array. (334). For example, the maximum and minimum thresholds provide upper and lower numbers of at least partially contiguous pixels that form a generally circular area representing the approximate geometric limits of specimen 1001 . Velocity and center position position records are tracked to verify movement of at least one specimen 1001 .

Algorithm 300 outputs the center position, velocity, and orientation data of specimen 1001 for further processing (336). For example, in some embodiments, algorithm 300 outputs data to display screen 110 (338) and to components of microcontroller 130 for viewing by an operator. In some embodiments, the algorithm 300 additionally outputs data to components of an external computing device via data connections. Once algorithm 300 has finished tracking specimen 1001 (340), algorithm 300 exits the processing flow loop. If algorithm 300 has not finished tracking specimen 1001 (340), algorithm 300 returns to capturing the first subsequent color image from the camera feed (308).

Specimens 1001 are exposed to a substantially constant centripetal force as programmed by the user, regardless of the axial position of the specimen 1001 within the specimen container 1000 (eg, the radial position of the specimen 1001 along the platform 104). Additionally, using information from algorithm 300 , microcontroller 130 may control the rotational speed, spin direction, and acceleration of platform 104 through communication with motor assembly 140 . For example, according to one or more signals transmitted by the microcontroller 130, the sample exerts sufficient centripetal force to move the sample 1001 along the central axis 1028 of the sample container 1000 toward the distal closure 1006 according to a specified protocol. To apply 1001 , platform 104 may spin about central axis 152 . The one or more signals may be used to adjust the angular velocity of platform 104 and/or the duration of one or more phases of the protocol. Such protocol adjustments may optimize the sample exposure time to the process medium within sample container 1000 .

In some embodiments, the sample container itself, otherwise similar to sample container 1000, may include one or more embedded optical elements (e.g., one or more lenses); The elements may be used to make the specimen 1001 more clearly visible to the naked eye or visualized by the camera 180 of the vision system 200 during an automated specimen tracking routine taking place in the specimen processing system 100 or separately. It is an element that

In some embodiments, the sample processing system 100 is designed to excite movement of either the sample 1001 or the fluid within the sample container 1000 or both while the sample container 1000 is being processed in the sample processing system 100. and further comprising one or more vibrating assemblies. For example, FIG. 20 illustrates such a vibration assembly 400 designed to securely support a specimen container 1000. As shown in FIG. Each of the one or more vibrating assemblies 400 may be installed in one or more of the processing stations 102 of the sample processing system 100 in place of the respective upper brackets 160 of the processing stations 102 .

Vibration assembly 400 includes a base 402 that can be secured to platform 104 at processing station 102 and to which other components of vibration assembly 400 are mounted. Vibration assembly 400 further includes a mounting platform 404 configured to support specimen container 1000 and movable (eg, suspended in free space) relative to base 402 . For example, the vibratory assembly 400 includes two frames 406 along which the mounting platform 404 can move laterally and longitudinally; two dynamic spacers that limit excessive outward movement due to centripetal force during spinning; 408 (eg, a spring or other member made of a compliant material); and an adjustable stop 410 that allows some free movement for the dynamic spacer 408 without having to rigidly attach the mounting platform 404 to the base 402. further includes At least the central portion of mounting platform 404 is made of an optically transparent material to allow focused light from vision system 200 to pass through and illuminate specimen 1001 within specimen container 1000 . Vibration assembly 400 also includes opposing restraining clamps 412 that can clamp specimen container 1000 to mounting platform 404 .

Vibration assembly 400 further includes motor 414 to vibrate mounting platform 404 along the x-axis and motor 416 to vibrate mounting platform 404 along the y-axis. Motors 414 , 416 may be activated via electrical signals received from electrical contacts 156 within housing 106 . Motors 414, 416 may be activated at the same time or at different times to achieve the desired direction of motion. The drive voltages of the motors 414,416 may also be adjusted to change the vibration frequency of the motors 414,416.

In some embodiments, sample processing systems that are similar in construction and function to sample processing system 100 may further include features for cutting and subsequently sealing sample containers 1000 . For example, in some embodiments, it may be necessary to cut excess length from sample container 1000 after sample 1001 has been processed and placed at the distal end of sample container 1000 . FIG. 21 illustrates the cut and seal station 502 of the sample processing system 500; here the sample container 1000 is simultaneously cut and sealed before the distal storage portion 503 of the sample container 1000 is placed in the cryogenic material 501 ( via heat sealing, ultrasonic sealing, or crimping). In some embodiments, specimen container 1000 may be cut and sealed in two separate operations. For example, FIG. 22 illustrates the cutting station 602 of the sample processing system 600, where the sample container 1000 may first be cut before the distal storage portion 603 of the sample container 1000 is placed in the cryogenic material 601. , and then may be automatically sealed, capped, or plugged using dedicated equipment that is part of the sample processing system 600 .

The sample processing system 100, sample processing system 500, sample processing system 600, sample container 1000, vision system 200, and vibration assembly 400 described above include components with certain dimensions, sizes, shapes, materials, and configurations. Although described and illustrated as such and with respect to the software algorithm 300, in some embodiments a sample processing system, sample container, vision system, which is otherwise substantially similar in structure and function to the embodiments described above. The vibrating assembly and software algorithms may include one or more components with different dimensions, sizes, shapes, materials and configurations, or one or more different process flow steps.

For example, although the sample processing system 100, vision system 200, and algorithm 300 have been described and illustrated with respect to tracking a single sample 1001 within a sample container 1000, in some embodiments the sample processing system is A sample processing system substantially similar to 100 may be operated with algorithms designed to track more than one sample 1001 within the same sample container 1000 during a sample processing protocol.

Accordingly, other aspects are within the scope of the appended claims.

Claims (40)

a plate for supporting a sample system comprising a container and a sample contained therein;
a camera disposed above the plate and configured to generate an image of the analyte system;
a light source positioned below the plate for directing light toward the plate;
a light stop for blocking a portion of the light from reaching the specimen system to provide darkfield illumination of the specimen at the camera;
and one or more processors electronically coupled to the camera and configured to track the position of the specimen within a specimen container during a specimen processing protocol based on the images. 11. The sample processing system of Claim 1, further comprising an adjustable lens for focusing light onto the sample system. 3. The sample processing system of Claim 1, further comprising a processing station for positioning said camera. 4. The sample processing system of Claim 3, wherein the processing station defines a receptacle adjacent the plate for positioning a sample container. 4. The specimen processing system of Claim 3, wherein the processing station comprises a mount for selectively positioning a camera at the processing station. 4. The sample processing system of Claim 3, further comprising a rotatable platform to which the processing station is fixed for applying a centripetal force to the sample to induce motion within the sample container. 7. The specimen processing system of Claim 6, wherein the one or more processors are further configured to convert an image from color to grayscale. 7. The specimen processing system of claim 6, wherein the one or more processors are further configured to remove noise from images. 7. The specimen processing system of claim 6, wherein the one or more processors are further configured to detect objects corresponding to specimens in images. 7. The one or more processors of claim 6, wherein the one or more processors are further configured to determine parameters including one or more of position, velocity, and direction of the specimen as it moves within the specimen container. Specimen processing system. 11. The sample processing system of Claim 10, wherein said one or more processors are configured to output one or more of said parameters. 12. The sample processing system of Claim 11, comprising a motor capable of adjusting movement of the rotatable platform based on one or more of said parameters. A light block is arranged to block a portion of the light from reaching the central axis of the sample container so that the edge of the sample remains visible to provide darkfield illumination at the camera. Item 1. The sample processing system according to item 1. 2. The sample processing system of Claim 1, wherein the light source comprises a plurality of light emitting diodes. 2. The sample processing system of Claim 1, wherein the camera is configured to scan an identification label on a sample container. 2. The sample processing system of claim 1, wherein the one or more processors are configured to track positions of each of a plurality of samples within a sample container based on images during a sample processing protocol. 3. The sample processing system of claim 1, further comprising a vibrating assembly configured to direct movement of the sample within the sample container during a sample processing protocol. 3. The sample processing system of claim 1, further comprising a cutting station configured to cut and release the distal portion of the sample container with the sample contained therein after completion of the sample processing protocol. 11. The specimen processing system of Claim 1, wherein the specimen comprises a reproductive specimen. 20. The sample processing system of Claim 19, wherein the sample processing protocol comprises a vitrification protocol. A method of processing a sample in a sample container, the method comprising the steps of:
generating an image of the specimen in the specimen container with a camera positioned above a plate supporting the specimen container;
directing light towards the plate from a light source positioned below the plate;
blocking a portion of the light from reaching the specimen with a light shield to provide darkfield illumination of the specimen at the camera; and one in electronic communication with the camera during a specimen processing protocol. or tracking, in a plurality of processors, the position of the specimen within the specimen container based on the image; 22. The method of Claim 21, further comprising focusing the light onto the specimen with an adjustable lens. 22. The method of claim 21, further comprising positioning a camera at a processing station. 24. The method of Claim 23, further comprising positioning a specimen container within a receptacle of a processing station adjacent said plate. 24. The method of Claim 23, further comprising selectively positioning a mount that supports the camera at the processing station. 24. The method of claim 23, further comprising applying a centripetal force to the specimen to induce movement in the specimen within the specimen container by rotating a platform to which the processing station is fixed. 27. The method of claim 26, further comprising converting an image from color to grayscale at the one or more processors. 27. The method of Claim 26, further comprising removing noise from the image at the one or more processors. 27. The method of claim 26, further comprising detecting objects corresponding to analytes in images at the one or more processors. 27. The method of claim 26, further comprising determining, at the one or more processors, parameters including one or more of position, velocity, and direction of the specimen as it moves within the specimen container. Method. 31. The method of claim 30, further comprising outputting one or more of said parameters from said one or more processors. 32. The method of claim 31, wherein the motors adjust the movement of the platform based on one or more of said parameters. 22. The method of claim 21, further comprising blocking a portion of the light from reaching the central axis of the specimen container so that the edges of the specimen remain visible to provide darkfield illumination at the camera. Method. 22. The method of Claim 21, wherein said light source comprises a plurality of light emitting diodes. 22. The method of claim 21, further comprising scanning an identification label of a specimen container with the camera. 22. The method of claim 21, further comprising tracking, at the one or more processors, a position of each of a plurality of specimens within a specimen container based on the images during a specimen processing protocol. 22. The method of claim 21, further comprising directing movement of the specimen within the specimen container with a vibrating assembly during a specimen processing protocol. 22. The method of claim 21, further comprising cutting and releasing the distal portion of the sample container with the sample contained therein at a cutting station after completion of the sample processing protocol. 22. The method of claim 21, wherein the specimen comprises a reproductive specimen. 40. The method of Claim 39, wherein the specimen processing protocol comprises a vitrification protocol.

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